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1.  Hydroxylation of recombinant human collagen type I alpha 1 in transgenic maize co-expressed with a recombinant human prolyl 4-hydroxylase 
BMC Biotechnology  2011;11:69.
Collagens require the hydroxylation of proline (Pro) residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H) as a posttranslational processing step.
A recombinant human collagen type I α-1 (rCIα1) with high percentage of hydroxylated prolines (Hyp) was produced in transgenic maize seeds when co-expressed with both the α- and β- subunits of a recombinant human P4H (rP4H). Germ-specific expression of rCIα1 using maize globulin-1 gene promoter resulted in an average yield of 12 mg/kg seed for the full-length rCIα1 in seeds without co-expression of rP4H and 4 mg/kg seed for the rCIα1 (rCIα1-OH) in seeds with co-expression of rP4H. High-resolution mass spectrometry (HRMS) analysis revealed that nearly half of the collagenous repeating triplets in rCIα1 isolated from rP4H co-expressing maize line had the Pro residues changed to Hyp residues. The HRMS analysis determined the Hyp content of maize-derived rCIα1-OH as 18.11%, which is comparable to the Hyp level of yeast-derived rCIα1-OH (17.47%) and the native human CIa1 (14.59%), respectively. The increased Hyp percentage was correlated with a markedly enhanced thermal stability of maize-derived rCIα1-OH when compared to the non-hydroxylated rCIα1.
This work shows that maize has potential to produce adequately modified exogenous proteins with mammalian-like post-translational modifications that may be require for their use as pharmaceutical and industrial products.
PMCID: PMC3151215  PMID: 21702901
2.  Abnormal Type I Collagen Post-translational Modification and Crosslinking in a Cyclophilin B KO Mouse Model of Recessive Osteogenesis Imperfecta 
PLoS Genetics  2014;10(6):e1004465.
Cyclophilin B (CyPB), encoded by PPIB, is an ER-resident peptidyl-prolyl cis-trans isomerase (PPIase) that functions independently and as a component of the collagen prolyl 3-hydroxylation complex. CyPB is proposed to be the major PPIase catalyzing the rate-limiting step in collagen folding. Mutations in PPIB cause recessively inherited osteogenesis imperfecta type IX, a moderately severe to lethal bone dysplasia. To investigate the role of CyPB in collagen folding and post-translational modifications, we generated Ppib−/− mice that recapitulate the OI phenotype. Knock-out (KO) mice are small, with reduced femoral areal bone mineral density (aBMD), bone volume per total volume (BV/TV) and mechanical properties, as well as increased femoral brittleness. Ppib transcripts are absent in skin, fibroblasts, femora and calvarial osteoblasts, and CyPB is absent from KO osteoblasts and fibroblasts on western blots. Only residual (2–11%) collagen prolyl 3-hydroxylation is detectable in KO cells and tissues. Collagen folds more slowly in the absence of CyPB, supporting its rate-limiting role in folding. However, treatment of KO cells with cyclosporine A causes further delay in folding, indicating the potential existence of another collagen PPIase. We confirmed and extended the reported role of CyPB in supporting collagen lysyl hydroxylase (LH1) activity. Ppib−/− fibroblast and osteoblast collagen has normal total lysyl hydroxylation, while increased collagen diglycosylation is observed. Liquid chromatography/mass spectrometry (LC/MS) analysis of bone and osteoblast type I collagen revealed site-specific alterations of helical lysine hydroxylation, in particular, significantly reduced hydroxylation of helical crosslinking residue K87. Consequently, underhydroxylated forms of di- and trivalent crosslinks are strikingly increased in KO bone, leading to increased total crosslinks and decreased helical hydroxylysine- to lysine-derived crosslink ratios. The altered crosslink pattern was associated with decreased collagen deposition into matrix in culture, altered fibril structure in tissue, and reduced bone strength. These studies demonstrate novel consequences of the indirect regulatory effect of CyPB on collagen hydroxylation, impacting collagen glycosylation, crosslinking and fibrillogenesis, which contribute to maintaining bone mechanical properties.
Author Summary
Osteogenesis imperfecta (OI), or brittle bone disease, is characterized by susceptibility to fractures from minimal trauma and growth deficiency. Deficiency of components of the collagen prolyl 3-hydroxylation complex, CRTAP, P3H1 and CyPB, cause recessive types VII, VIII and IX OI, respectively. We have previously shown that mutual protection within the endoplasmic reticulum accounts for the overlapping severe phenotype of patients with CRTAP and P3H1 mutations. However, the bone dysplasia in patients with CyPB deficiency is distinct in terms of phenotype and type I collagen biochemistry. Using a knock-out mouse model of type IX OI, we have demonstrated that CyPB is the major, although not unique, peptidyl prolyl cis-trans isomerase that catalyzes the rate-limiting step in collagen folding. CyPB is also required for activity of the collagen prolyl 3-hydroxylation complex; collagen α1(I) P986 modification is lost in the absence of CyPB. Unexpectedly, CyPB was found to also influence collagen helical lysyl hydroxylation in a tissue-, cell- and residue-specific manner. Thus CyPB facilitates collagen folding directly, but also indirectly regulates collagen hydroxylation, glycosylation, crosslinking and fibrillogenesis through its interactions with other collagen modifying enzymes in the endoplasmic reticulum.
PMCID: PMC4072593  PMID: 24968150
3.  Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues 
PLoS Genetics  2014;10(1):e1004121.
Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex's 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1H662A). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.
Author Summary
The prolyl 3-hydroxylase complex serves to hydroxylate a single residue in type I collagen and also serves as a collagen chaperone. The complex is comprised of prolyl 3-hydroxylase 1, cartilage associated protein, and cyclophilin B. Mutations have been identified in the genes encoding the complex members in patients with recessive Osteogenesis Imperfecta. Recent data suggest that prolyl 3-hydroxylation of collagen does not alter the stability of collagen but may rather mediate protein-protein interactions. Additionally, the collagen chaperoning function of the complex is an important rate limiting step in the modification of type I collagen. Irrespective of whether patients with mutations in the genes encoding the members of the prolyl 3-hydroxylase complex have hypomorphic or complete loss of function alleles, either circumstance leads to the loss of both functions of the prolyl 3-hydroxylase complex. Thus, it is unknown how collagen chaperoning and/or hydroxylation affect bone and cartilage homeostasis. In this study, we generated a mouse model lacking the prolyl 3-hydroxylation activity of the complex while maintaining the chaperoning function. We found that the hydroxylase mutant mice have normal cartilage and normal cortical bone but decreased trabecular bone, suggesting that there is a differential requirement for hydroxylation in different tissues.
PMCID: PMC3900401  PMID: 24465224
4.  Tunable, post-translational hydroxylation of collagen domains in Escherichia coli 
ACS Chemical Biology  2011;6(4):320-324.
Prolyl 4-hydroxylases are ascorbate-dependent oxygenases that play key roles in a variety of eukaryotic biological processes including oxygen sensing, siRNA regulation, and collagen folding. They perform their functions by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S,4R)-4-hydroxyproline. Thus far, our ability to study these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. By introducing a biosynthetic shunt to produce ascorbate-like molecules in Eschericia coli cells that heterologously express human prolyl 4-hydroxylase (P4H), we have created a strain of Escherichia coli that produces collagenous proteins with high levels of (2S,4R)-4-hydroxyproline. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology, and create a foundation for better understanding how post-translational hydroxylation affects proteins.
PMCID: PMC3337207  PMID: 21210682
5.  Prolyl 4-Hydroxylase Is an Essential Procollagen-Modifying Enzyme Required for Exoskeleton Formation and the Maintenance of Body Shape in the Nematode Caenorhabditis elegans 
Molecular and Cellular Biology  2000;20(11):4084-4093.
The multienzyme complex prolyl 4-hydroxylase catalyzes the hydroxylation of proline residues and acts as a chaperone during collagen synthesis in multicellular organisms. The β subunit of this complex is identical to protein disulfide isomerase (PDI). The free-living nematode Caenorhabditis elegans is encased in a collagenous exoskeleton and represents an excellent model for the study of collagen biosynthesis and extracellular matrix formation. In this study, we examined prolyl 4-hydroxylase α-subunit (PHY; EC and β-subunit (PDI; EC genes with respect to their role in collagen modification and formation of the C. elegans exoskeleton. We identified genes encoding two PHYs and a single associated PDI and showed that all three are expressed in collagen-synthesizing ectodermal cells at times of maximal collagen synthesis. Disruption of the pdi gene via RNA interference resulted in embryonic lethality. Similarly, the combined phy genes are required for embryonic development. Interference with phy-1 resulted in a morphologically dumpy phenotype, which we determined to be identical to the uncharacterized dpy-18 locus. Two dpy-18 mutant strains were shown to have null alleles for phy-1 and to have a reduced hydroxyproline content in their exoskeleton collagens. This study demonstrates in vivo that this enzyme complex plays a central role in extracellular matrix formation and is essential for normal metazoan development.
PMCID: PMC85778  PMID: 10805750
6.  Collagen prolyl3-hydroxylation: a major role for a minor post-translational modification? 
Connective tissue research  2013;54(0):245-251.
Prolyl 3-hydroxylation is a rare but conserved post-translational modification in many collagen types and, when defective, may be linked to a number of human diseases with musculoskeletal and potentially ocular and renal pathologies. Prolyl 3-hydroxylase-1 (P3H1), the enzyme responsible for converting proline to 3-hydroxyproline (3Hyp) in type I collagen, requires the coenzyme CRTAP for activity. Mass spectrometric analysis showed that the Crtap−/− mouse was missing 3-hydroxyproline in type I collagen α-chains. This finding led to the discovery mutations in genes encoding the P3H1 complex as a cause of recessively inherited osteogenesis imperfecta (brittle bone disease). Since then, many additional 3Hyp sites have been identified in various collagen types and classified based on observed substrate and tissue specificity. P3H1 is part of a family of gene products that also includes isoenzymes P3H2 and P3H3 as well as CRTAP and Sc65. It is believed these isoenzymes and coenzymes have evolved different collagen substrate site and tissue specificities in their activities. The post-translational fingerprinting of collagens will be essential in understanding the basic role and extent of regulated variations of prolyl 3-hydroxylation in collagen. We believe that prolyl 3-hydroxylation is a functionally significant collagen post-translational modification and can be a cause of disease when absent.
PMCID: PMC3995746  PMID: 23772978
3-hydroxyproline; fibrillar collagen; post-translational modification; prolyl 3-hydroxylase
7.  The Aberrance of the 4S Diastereomer of 4-Hydroxyproline 
Journal of the American Chemical Society  2010;132(31):10857-10865.
Prolyl 4-hydroxylases install a hydroxyl group in the 4R configuration on the γ-carbon atom of certain (2S)-proline (Pro) residues in tropocollagen, elastin, and other proteins and other proteins to form (2S,4R)-4-hydroxyproline (Hyp). The gauche effect arising from this prevalent post-translational modification enforces a Cγ-exo ring pucker and stabilizes the collagen triple helix. The Hyp diastereomer (2S,4S)-4-hydroxyproline (hyp) has not been observed in a protein, despite the ability of electronegative 4S substituents to enforce the more common Cγ-endo ring pucker of Pro. Here, we use density functional theory, spectroscopy, crystallography, and calorimetry to explore the consequences of hyp incorporation on protein stability using a collagen model system. We find that the 4S-hydroxylation of Pro to form hyp does indeed enforce a Cγ-endo ring pucker, but a transannular hydrogen bond between the hydroxyl moiety and the carbonyl of hyp distorts the main-chain torsion angles that typically accompany a Cγ-endo ring pucker. This same transannular hydrogen bond enhances an n→π* interaction that stabilizes the trans conformation of the peptide bond preceding hyp, endowing hyp with the unusual combination of a Cγ-endo ring pucker and high trans:cis ratio. O-Methylation of hyp to form (2S,4S)-4-methoxyproline (mop) eliminates the transannular hydrogen bond and restores a prototypical Cγ-endo pucker. mop residues endow the collagen triple helix with much more conformational stability than do hyp residues. These findings highlight the critical importance of the configuration of the hydroxyl group installed on Cγ of proline residues.
PMCID: PMC2931826  PMID: 20681719
8.  Generalized Connective Tissue Disease in Crtap-/- Mouse 
PLoS ONE  2010;5(5):e10560.
Mutations in CRTAP (coding for cartilage-associated protein), LEPRE1 (coding for prolyl 3-hydroxylase 1 [P3H1]) or PPIB (coding for Cyclophilin B [CYPB]) cause recessive forms of osteogenesis imperfecta and loss or decrease of type I collagen prolyl 3-hydroxylation. A comprehensive analysis of the phenotype of the Crtap-/- mice revealed multiple abnormalities of connective tissue, including in the lungs, kidneys, and skin, consistent with systemic dysregulation of collagen homeostasis within the extracellular matrix. Both Crtap-/- lung and kidney glomeruli showed increased cellular proliferation. Histologically, the lungs showed increased alveolar spacing, while the kidneys showed evidence of segmental glomerulosclerosis, with abnormal collagen deposition. The Crtap-/- skin had decreased mechanical integrity. In addition to the expected loss of proline 986 3-hydroxylation in α1(I) and α1(II) chains, there was also loss of 3Hyp at proline 986 in α2(V) chains. In contrast, at two of the known 3Hyp sites in α1(IV) chains from Crtap-/- kidneys there were normal levels of 3-hydroxylation. On a cellular level, loss of CRTAP in human OI fibroblasts led to a secondary loss of P3H1, and vice versa. These data suggest that both CRTAP and P3H1 are required to maintain a stable complex that 3-hydroxylates canonical proline sites within clade A (types I, II, and V) collagen chains. Loss of this activity leads to a multi-systemic connective tissue disease that affects bone, cartilage, lung, kidney, and skin.
PMCID: PMC2868021  PMID: 20485499
9.  Absence of FKBP10 in Recessive Type XI Osteogenesis Imperfecta Leads to Diminished Collagen Cross-Linking and Reduced Collagen Deposition in Extracellular Matrix 
Human mutation  2012;33(11):1589-1598.
Recessive osteogenesis imperfecta (OI) is caused by defects in genes whose products interact with type I collagen for modification and/or folding. We identified a Palestinian pedigree with moderate and lethal forms of recessive OI caused by mutations in FKBP10 or PPIB, which encode endoplasmic reticulum resident chaperone/isomerases FKBP65 and CyPB, respectively. In one pedigree branch, both parents carry a deletion in PPIB (c.563_566delACAG), causing lethal type IX OI in their two children. In another branch, a child with moderate type XI OI has a homozygous FKBP10 mutation (c.1271_1272delCCinsA). Proband FKBP10 transcripts are 4% of control and FKBP65 protein is absent from proband cells. Proband collagen electrophoresis reveals slight band broadening, compatible with ≈10% overmodification. Normal chain incorporation, helix folding, and collagen Tm support a minimal general collagen chaperone role for FKBP65. However, there is a dramatic decrease in collagen deposited in culture despite normal collagen secretion. Mass spectrometry reveals absence of hydroxylation of the collagen telopeptide lysine involved in cross-linking, suggesting that FKBP65 is required for lysyl hydroxylase activity or access to type I collagen telopeptide lysines, perhaps through its function as a peptidylprolyl isomerase. Proband collagen to organics ratio in matrix is approximately 30% of normal in Raman spectra. Immunofluorescence shows sparse, disorganized collagen fibrils in proband matrix.
PMCID: PMC3470738  PMID: 22718341
osteogenesis imperfecta; Bruck syndrome; FKBP65; FKBP10; PPIB; peptidylprolyl isomerase
10.  Evolutionary Origins of C-Terminal (GPP)n 3-Hydroxyproline Formation in Vertebrate Tendon Collagen 
PLoS ONE  2014;9(4):e93467.
Approximately half the proline residues in fibrillar collagen are hydroxylated. The predominant form is 4-hydroxyproline, which helps fold and stabilize the triple helix. A minor form, 3-hydroxyproline, still has no clear function. Using peptide mass spectrometry, we recently revealed several previously unknown molecular sites of 3-hydroxyproline in fibrillar collagen chains. In fibril-forming A-clade collagen chains, four new partially occupied 3-hydroxyproline sites were found (A2, A3, A4 and (GPP)n) in addition to the fully occupied A1 site at Pro986. The C-terminal (GPP)n motif has five consecutive GPP triplets in α1(I), four in α2(I) and three in α1(II), all subject to 3-hydroxylation. The evolutionary origins of this substrate sequence were investigated by surveying the pattern of its 3-hydroxyproline occupancy from early chordates through amphibians, birds and mammals. Different tissue sources of type I collagen (tendon, bone and skin) and type II collagen (cartilage and notochord) were examined by mass spectrometry. The (GPP)n domain was found to be a major substrate for 3-hydroxylation only in vertebrate fibrillar collagens. In higher vertebrates (mouse, bovine and human), up to five 3-hydroxyproline residues per (GPP)n motif were found in α1(I) and four in α2(I), with an average of two residues per chain. In vertebrate type I collagen the modification exhibited clear tissue specificity, with 3-hydroxyproline prominent only in tendon. The occupancy also showed developmental changes in Achilles tendon, with increasing 3-hydroxyproline levels with age. The biological significance is unclear but the level of 3-hydroxylation at the (GPP)n site appears to have increased as tendons evolved and shows both tendon type and developmental variations within a species.
PMCID: PMC3973637  PMID: 24695516
11.  Proline-Hydroxylated Hypoxia-Inducible Factor 1α (HIF-1α) Upregulation in Human Tumours 
PLoS ONE  2014;9(2):e88955.
The stabilisation of HIF-α is central to the transcriptional response of animals to hypoxia, regulating the expression of hundreds of genes including those involved in angiogenesis, metabolism and metastasis. HIF-α is degraded under normoxic conditions by proline hydroxylation, which allows for recognition and ubiquitination by the von-Hippel-Lindau (VHL) E3 ligase complex. The aim of our study was to investigate the posttranslational modification of HIF-1α in tumours, to assess whether there are additional mechanisms besides reduced hydroxylation leading to stability. To this end we optimised antibodies against the proline-hydroxylated forms of HIF-1α for use in formalin fixed paraffin embedded (FFPE) immunohistochemistry to assess effects in tumour cells in vivo. We found that HIF-1α proline-hydroxylated at both VHL binding sites (Pro402 and Pro564), was present in hypoxic regions of a wide range of tumours, tumour xenografts and in moderately hypoxic cells in vitro. Staining for hydroxylated HIF-1α can identify a subset of breast cancer patients with poorer prognosis and may be a better marker than total HIF-1α levels. The expression of unhydroxylated HIF-1α positively correlates with VHL in breast cancer suggesting that VHL may be rate-limiting for HIF degradation. Our conclusions are that the degradation of proline-hydroxylated HIF-1α may be rate-limited in tumours and therefore provides new insights into mechanisms of HIF upregulation. Persistence of proline-hydroxylated HIF-1α in perinecrotic areas suggests there is adequate oxygen to support prolyl hydroxylase domain (PHD) activity and proline-hydroxylated HIF-1α may be the predominant form associated with the poorer prognosis that higher levels of HIF-1α confer.
PMCID: PMC3923075  PMID: 24563687
12.  The collagen prolyl hydroxylases are novel transcriptionally silenced genes in lymphoma 
British Journal of Cancer  2012;107(8):1423-1432.
Prolyl hydroxylation is a post-translational modification that affects the structure, stability and function of proteins including collagen by catalysing hydroxylation of proline to hydroxyproline through action of collagen prolyl hydroxylases3 (C-P3H) and 4 (C-P4H). Three C-P3Hs (nomenclature was amended according to approval by the HGNC symbols and names at ( and Entrez database at ( leucineproline-enriched proteoglycan (leprecan) 1 (Lepre1), leprecan-like 1 (Leprel1), leprecan-like 2 (Leprel2) and two paralogs Cartilage-Related Protein (CRTAP) and leprecan-like 4 (Leprel4) are found in humans. The C-P4Hs are tetrameric proteins comprising a variable α subunit, encoded by the P4HA1, P4HA2 and P4HA3 genes and a constant β subunit encoded by P4HB.
We used RT–PCR, qPCR, pyrosequencing, methylation-specific PCR, western blotting and immunohistochemistry to investigate expression and regulation of the C-P3H and C-P4H genes in B lymphomas and normal bone marrow.
C-P3H and C-P4H are downregulated in lymphoma. Down-regulation is associated with methylation in the CpG islands and is detected in almost all common types of B-cell lymphoma, but the CpG islands are unmethylated or methylated at lower levels in DNA isolated from normal bone marrow and lymphoblastoid cell lines. Methylation of multiple C-P3H and C-P4H genes is present in some lymphomas, particularly Burkitt's lymphoma.
Methylation of C-P3H and C-P4H is common in B lymphomas and may have utility in differentiating disease subtypes.
PMCID: PMC3494450  PMID: 22955849
non-Hodgkin lymphoma; Prolyl hydroxylases; methylation; epigenetics
13.  Supramolecular assembly of electrostatically stabilized, hydroxyproline-lacking collagen-mimetic peptides 
Biomacromolecules  2009;10(9):2626-2631.
The mechanical and biological functions of the native collagens remain an inspiration in materials design, but widespread application of de novo collagens has been limited in part by the need for hydroxylated proline in the formation of stable triple helical structures. In order to address this continued need and to expand the potential for recombinant expression of functional, hydroxyproline-lacking collagen-mimetic peptides, we have designed a hydrophilic, non-repetitive, and thermally stable collagen-mimetic peptide via the incorporation of triple-helix-stabilizing charged triplets. The peptide sequence is also equipped with a type III-collagen-mimetic cystine knot at the C-terminus to facilitate covalent crosslinking of the triple helix via simple air oxidation. Circular dichroic (CD) studies of this collagen-mimetic peptide revealed a typical, thermally stable, collagen triple helix signature, with a weak positive maximum at 225 nm, and a triple helix melting temperature (Tm) of 35 °C and 43 °C for the reduced and oxidized forms respectively. The thermal behavior was confirmed via analysis by differential scanning calorimetry. Interestingly, this hydroxyproline-lacking, collagen-mimetic peptide also assembles into nanorods and microfibrillar structures as observed via transmission electron microscopy. The identification and demonstrated useful collagen-mimetic properties of this peptide suggests important opportunities in the recombinant design of new collagen-based biomaterials.
PMCID: PMC2751732  PMID: 19681603
Collagen peptide; collagen triple helix; self-assembly; nanorods; microfibrils
14.  Peptidyl 3-Hydroxyproline-Binding Properties of Type I Collagen Suggest a Function in Fibril Supramolecular Assembly 
Biochemistry  2012;51(12):2417-2424.
Proline residues in collagens are extensively hydroxylated post-translationally. A rare form of this modification, 3S, 2S-L-hydroxyproline (3Hyp), remains without a clear function. Disruption of the enzyme complex responsible for prolyl 3-hydroxylation results in severe forms of recessive osteogenesis imperfecta (OI). These OI types exhibit a loss or reduction of 3-hydroxylation at two proline residues, α1(I) Pro986 and α2(I) Pro707. Whether the resulting brittle bone phenotype is caused by the lack of the 3-hydroxyl addition or by another function of the enzyme complex is unknown. We have speculated that the most efficient mechanism to explain the chemistry of collagen intermolecular cross-linking is for pairs of collagen molecules in register to be the subunit that assembles into fibrils. In this concept the exposed hydroxyls from 3Hyp are positioned within mutually interactive binding motifs on adjacent collagen molecules that contribute through hydrogen bonding to the process of fibril supramolecular assembly. Here we report observations on the physical binding properties of 3Hyp in collagen chains from experiments designed to explore the potential for interaction using synthetic collagen-like peptides containing 3Hyp. Evidence of self-association was observed between a synthetic peptide containing 3Hyp and the CB6 domain of the α1(I) chain, which contains the single fully 3-hydroxylated proline. Using collagen from a case of severe recessive OI with a CRTAP defect, in which Pro986 was minimally 3-hydroxylated, such binding was not observed. Further study on the role of 3Hyp in supramolecular assembly is warranted for understanding the evolution of tissue-specific variations in collagen fibril organization.
PMCID: PMC3314591  PMID: 22380708
collagen; 3-hydroxyproline; bone; supramolecular assembly; osteogenesis imperfecta
15.  Direct and continuous assay for prolyl 4-hydroxylase 
Analytical biochemistry  2008;386(2):181-185.
Prolyl 4-hydroxylase (P4H) is a non-heme iron dioxygenase that catalyzes the post-translational hydroxylation of (2S)-proline (Pro) residues in protocollagen strands. The resulting (2S,4R)-4-hydroxyproline (Hyp) residues are essential for the folding, secretion, and stability of the collagen triple helix. P4H uses α-ketoglutarate and O2 as co-substrates, and forms succinate and CO2 as well as Hyp. Described herein is the first assay for P4H that continuously and directly detects turnover of the proline-containing substrate. This assay is based on (2S,4S)-4-fluoroproline (flp), a proline analogue that is transformed into (2S)-4-ketoproline (Kep) and inorganic fluoride by P4H. The fluoride ion, and thus turnover by P4H, is detected by a fluoride ion-selective electrode. Using this assay, steady-state kinetic parameters for the human P4H-catalyzed turnover of a flp-containing peptide were determined and found to be comparable to those obtained with a discontinuous HPLC-based assay. In addition, this assay can be used to characterize P4H variants, as demonstrated by a comparison of catalysis by D414A P4H and the wild-type enzyme. Finally, the use of the assay to identify small-molecule inhibitors of P4H was verified by an analysis of catalysis in the presence of 2,4-pyridine dicarboxylate, an analogue of α-ketoglutarate. Thus, the assay described herein could facilitate biochemical analyses of this essential enzyme.
PMCID: PMC2643311  PMID: 19111518
16.  Steroid conversion with CYP106A2 – production of pharmaceutically interesting DHEA metabolites 
Steroids are lipophilic compounds with a gonane skeleton and play an important role in higher organisms. Due to different functionalizations - mainly hydroxylations - at the steroid molecule, they vary highly in their mode of action. The pharmaceutical industry is, therefore, interested in hydroxysteroids as therapeutic agents. The insertion of hydroxyl groups into a steroid core, however, is hardly accomplishable by classical chemical means; that is because microbial steroid hydroxylations are investigated and applied since decades. CYP106A2 is a cytochrome P450 monooxygenase from Bacillus megaterium ATCC 13368, which was first described in the late 1970s and which is capable to hydroxylate a variety of 3-oxo-delta4 steroids at position 15beta. CYP106A2 is a soluble protein, easy to express and to purify in high amounts, which makes this enzyme an interesting target for biotechnological purposes.
In this work a focused steroid library was screened in vitro for new CYP106A2 substrates using a reconstituted enzyme assay. Five new substrates were identified, including dehydroepiandrosterone and pregnenolone. NMR-spectroscopy revealed that both steroids are mainly hydroxylated at position 7beta. In order to establish a biotechnological system for the preparative scale production of 7beta-hydroxylated dehydroepiandrosterone, whole-cell conversions with growing and resting cells of B. megaterium ATCC1336 the native host of CYP1062 and also with resting cells of a recombinant B. megaterium MS941 strain overexpressing CYP106A2 have been conducted and conversion rates of 400 muM/h (115 mg/l/h) were obtained. Using the B. megaterium MS941 overexpression strain, the selectivity of the reaction was improved from 0.7 to 0.9 for 7beta-OH-DHEA.
In this work we describe CYP106A2 for the first time as a regio-selective hydroxylase for 3-hydroxy-delta5 steroids. DHEA was shown to be converted to 7beta-OH-DHEA which is a highly interesting human metabolite, supposed to act as neuroprotective, anti-inflammatory and immune-modulatory agent. Optimization of the whole-cell system using different B. megaterium strains lead to a conversion of DHEA with B. megaterium showing high selectivity and conversion rates and displaying a volumetric yield of 103 mg/l/h 7beta-OH-DHEA.
PMCID: PMC4080778  PMID: 24903845
Cytochrome P450; Steroid hydroxylase; Bacillus megaterium; Whole-cell conversion; Dehydroepiandrosterone; Microbial; CYP106A2
17.  Requirements for Skp1 processing by cytosolic prolyl 4(trans)-hydroxylase and α-N-acetylglucosaminyltransferase enzymes involved in O2-signaling in Dictyostelium† 
Biochemistry  2011;50(10):1700-1713.
The social amoeba Dictyostelium expresses a hypoxia inducible factor-α (HIFα)-type prolyl 4-hydroxylase (P4H1) and an α-N-acetylglucosaminyltransferase (Gnt1) that sequentially modify proline-143 of Skp1, a subunit of the SCF (Skp1/Cullin/F-box protein)-class of E3 ubiquitin-ligases. Prior genetic studies have implicated Skp1 and its modification by these enzymes in O2-regulation of development, suggesting the existence of an ancient O2-sensing mechanism related to modification of the transcription factor HIFα by animal prolyl 4-hydroxylases (PHDs). To better understand the role of Skp1 in P4H1-dependent O2-signaling, biochemical and biophysical studies were conducted to characterize the reaction product and the basis of Skp1 substrate selection by P4H1 and Gnt1. 1H-NMR demonstrated formation of 4(trans)-hydroxyproline as previously found for HIFα, and highly purified P4H1 was inhibited by Krebs cycle intermediates and other compounds that affect animal P4Hs. However, in contrast to hydroxylation of HIFα by PHDs, P4H1 depended on features of full-length Skp1, based on truncation, mutagenesis, and competitive inhibition studies. These features are conserved during animal evolution, as even mammalian Skp1, which lacks the target proline, became a good substrate upon its restoration. P4H1 recognition may depend on features conserved for SCF complex formation as heterodimerization with an F-box protein blocked Skp1 hydroxylation. The hydroxyproline-capping enzyme Gnt1 exhibited similar requirements for Skp1 as a substrate. These and other findings support a model in which the protist P4H1 conditionally hydroxylates Skp1 of E3SCFubiquitin-ligases to control half-lives of multiple targets, rather than the mechanism of animal PHDs where individual proteins are hydroxylated leading to ubiquitination by the evolutionarily-related E3VBCubiquitin-ligases.
PMCID: PMC3192012  PMID: 21247092
4-hydroxyproline; cellular slime mold; cytoplasmic glycosylation; peptide NMR
18.  Proline Availability Regulates Proline-4-Hydroxylase Synthesis and Substrate Uptake in Proline-Hydroxylating Recombinant Escherichia coli 
Microbial physiology plays a crucial role in whole-cell biotransformation, especially for redox reactions that depend on carbon and energy metabolism. In this study, regio- and enantio-selective proline hydroxylation with recombinant Escherichia coli expressing proline-4-hydroxylase (P4H) was investigated with respect to its interconnectivity to microbial physiology and metabolism. P4H production was found to depend on extracellular proline availability and on codon usage. Medium supplementation with proline did not alter p4h mRNA levels, indicating that P4H production depends on the availability of charged prolyl-tRNAs. Increasing the intracellular levels of soluble P4H did not result in an increase in resting cell activities above a certain threshold (depending on growth and assay temperature). Activities up to 5-fold higher were reached with permeabilized cells, confirming that host physiology and not the intracellular level of active P4H determines the achievable whole-cell proline hydroxylation activity. Metabolic flux analysis revealed that tricarboxylic acid cycle fluxes in growing biocatalytically active cells were significantly higher than proline hydroxylation rates. Remarkably, a catalysis-induced reduction of substrate uptake was observed, which correlated with reduced transcription of putA and putP, encoding proline dehydrogenase and the major proline transporter, respectively. These results provide evidence for a strong interference of catalytic activity with the regulation of proline uptake and metabolism. In terms of whole-cell biocatalyst efficiency, proline uptake and competition of P4H with proline catabolism are considered the most critical factors.
PMCID: PMC3623152  PMID: 23455348
19.  Prolyl-4-hydroxylase α subunit 2 promotes breast cancer progression and metastasis by regulating collagen deposition 
BMC Cancer  2014;14:1.
Increased collagen deposition provides physical and biochemical signals to support tumor growth and invasion during breast cancer development. Therefore, inhibition of collagen synthesis and deposition has been considered a strategy to suppress breast cancer progression. Collagen prolyl-4-hydroxylase α subunit 2 (P4HA2), an enzyme hydroxylating proline residues in -X-Pro-Gly- sequences, is a potential therapeutic target for the disorders associated with increased collagen deposition. However, expression and function of P4HA2 in breast cancer progression are not well investigated.
Gene co-expression analysis was performed in the published microarray datasets to identify potential regulators of collagen I, III, and IV in human breast cancer tissue. Expression of P4HA2 was silenced by shRNAs, and its activity was inhibited by 1, 4-DPCA, a prolyl-4-hydroxylase inhibitor. Three-dimensional culture assay was used to analyze roles of P4HA2 in regulating malignant phenotypes of breast cancer cells. Reduced deposition of collagen I and IV was detected by Western blotting and immunofluorescence. Control and P4HA2-silenced breast cancer cells were injected into fat pad and tail vein of SCID mice to examine effect of P4HA2 on tumor growth and lung metastasis.
Using gene co-expression analysis, we showed that P4HA2 was associated with expression of Col1A1, Col3A1, and Col4A1 during breast cancer development and progression. P4HA2 mRNA levels were significantly upregulated in breast cancer compared to normal mammary tissue. Increased mRNA levels of P4HA2 correlated with poor clinical outcome in breast cancer patients, which is independent of estrogen receptor status. Silencing P4HA2 expression or treatment with the P4HA inhibitor significantly inhibited cell proliferation and suppressed aggressive phenotypes of breast cancer cells in 3D culture, accompanied by reduced deposition of collagen I and IV. We also found that knockdown of P4HA2 inhibited mammary tumor growth and metastasis to lungs in xenograft models.
These results suggest the critical role of P4HA2 in breast cancer progression and identify P4HA2 as a potential therapeutic target and biomarker for breast cancer progression.
PMCID: PMC3880410  PMID: 24383403
Tumor microenvironment; Breast cancer; Collagen deposition; Cancer progression; Cell proliferation
20.  Detection of Novel Proline 3-Hydroxylase Activities in Streptomyces and Bacillus spp. by Regio- and Stereospecific Hydroxylation of l-Proline 
During the screening of microbial proline hydroxylases, novel proline 3-hydroxylase activities, which hydroxylate free l-proline to free cis-3-hydroxy-l-proline, were detected in whole cells of Streptomyces sp. strain TH1 and Bacillus sp. strains TH2 and TH3 from 3,000 strains isolated from soil. The reaction product was purified from a reaction mixture of Streptomyces sp. strain TH1, and its chemical structure was identified as cis-3-hydroxy-l-proline by instrumental analyses. Proline 3-hydroxylase activity was also detected in Streptomyces canus ATCC 12647 which produces the 3-hydroxyproline-containing peptide antibiotic telomycin. Bacillus sp. strains TH2 and TH3 were found to accumulate cis-3-hydroxy-l-proline in culture media at 426 and 352 (mu)M, respectively. It was suggested that hydroxylation occurred in a highly regio- and stereospecific manner at position 3 of l-proline because no hydroxylation product other than cis-3-hydroxy-l-proline was observed. Proline 3-hydroxylases of these strains were first characterized on crude enzyme preparations. Since 2-oxoglutarate and ferrous ion were required for hydroxylation of l-proline, these 3-hydroxylases were thought to belong to a family of 2-oxoglutarate-related dioxygenases. The reaction was inhibited by Co(sup2+), Zn(sup2+), and Cu(sup2+). l-Ascorbic acid accelerated the reaction. The optimum pH and temperature were 7.5 and 35(deg)C, respectively.
PMCID: PMC1388867  PMID: 16535329
21.  Molecular characterization and functional expression of flavonol 6-hydroxylase 
BMC Plant Biology  2004;4:20.
Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis.
A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4'-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome.
Recombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction.
PMCID: PMC544895  PMID: 15596008
22.  Core Glycosylation of Collagen Is Initiated by Two β(1-O)Galactosyltransferases ▿ †  
Molecular and Cellular Biology  2008;29(4):943-952.
Collagen is a trimer of three left-handed alpha chains representing repeats of the motif Gly-X-Y, where (hydroxy)proline and (hydroxy)lysine residues are often found at positions X and Y. Selected hydroxylysines are further modified by the addition of galactose and glucose-galactose units. Collagen glycosylation takes place in the endoplasmic reticulum before triple-helix formation and is mediated by β(1-O)galactosyl- and α(1-2)glucosyltransferase enzymes. We have identified two collagen galactosyltransferases using affinity chromatography and tandem mass spectrometry protein sequencing. The two collagen β(1-O)galactosyltransferases corresponded to the GLT25D1 and GLT25D2 proteins. Recombinant GLT25D1 and GLT25D2 enzymes showed a strong galactosyltransferase activity toward various types of collagen and toward the serum mannose-binding lectin MBL, which contains a collagen domain. Amino acid analysis of the products of GLT25D1 and GLT25D2 reactions confirmed the transfer of galactose to hydroxylysine residues. The GLT25D1 gene is constitutively expressed in human tissues, whereas the GLT25D2 gene is expressed only at low levels in the nervous system. The GLT25D1 and GLT25D2 enzymes are similar to CEECAM1, to which we could not attribute any collagen galactosyltransferase activity. The GLT25D1 and GLT25D2 genes now allow addressing of the biological significance of collagen glycosylation and the importance of this posttranslational modification in the etiology of connective tissue disorders.
PMCID: PMC2643808  PMID: 19075007
23.  Extracellular Matrix Dynamics in Hepatocarcinogenesis: a Comparative Proteomics Study of PDGFC Transgenic and Pten Null Mouse Models 
PLoS Genetics  2011;7(6):e1002147.
We are reporting qualitative and quantitative changes of the extracellular matrix (ECM) and associated receptor proteomes, occurring during the transition from liver fibrosis and steatohepatitis to hepatocellular carcinoma (HCC). We compared two mouse models relevant to human HCC: PDGFC transgenic (Tg) and Pten null mice, models of disease progression from fibrosis and steatohepatitis to HCC. Using mass spectrometry, we identified in the liver of both models proteins for 26 collagen-encoding genes, providing the first evidence of expression at the protein level for 16 collagens. We also identified post-transcriptional protein variants for six collagens and lysine hydroxylation modifications for 14 collagens. Tumor-associated collagen proteomes were similar in both models with increased expression of collagens type IV, VI, VII, X, XIV, XV, XVI, and XVIII. Splice variants for Col4a2, Col6a2, Col6a3 were co-upregulated while only the short form of Col18a1 increased in the tumors. We also identified tumor specific increases of nidogen 1, decorin, perlecan, and of six laminin subunits. The changes in these non-collagenous ECM proteins were similar in both models with the exception of laminin β3, detected specifically in the Pten null tumors. Pdgfa and Pdgfc mRNA expression was increased in the Pten null liver, a possible mechanism for the similarity in ECM composition observed in the tumors of both models. In contrast and besides the strong up-regulation of integrin α5 protein observed in the liver tumors of both models, the expression of the six other integrins identified was specific to each model, with integrins α2b, α3, α6, and β1 up-regulated in Pten null tumors and integrins α8 and β5 up-regulated in the PDGFC Tg tumors. In conclusion, HCC–associated ECM proteins and ECM–integrin networks, common or specific to HCC subtypes, were identified, providing a unique foundation to using ECM composition for HCC classification, diagnosis, prevention, or treatment.
Author Summary
The microenvironment can have a profound influence on cellular behavior and survival and on growth of developing tumor cells. We present the first comprehensive analysis of the extracellular matrix (ECM) and associated receptor proteomes, applied here to the study of hepatocellular carcinoma (HCC). This study demonstrates the utility of mass spectrometry-based approaches to characterize, at the protein level, gene families with extensive sequence homology, post-transcriptional regulations, and post-translational regulations. This is also the first study to analyze and compare liver proteome changes occurring during the transition from fibrosis and steatohepatitis, common preneoplastic conditions in humans, to HCC, using two mouse models. This approach identifies ECM and integrin components, which could play an important role in the early steps of hepatocarcinogenesis, and provides a path to identifying ECM–tumor cell networks that may contribute to the heterogeneous features of HCC.
PMCID: PMC3121762  PMID: 21731504
24.  Microbial Proline 4-Hydroxylase Screening and Gene Cloning 
Microbial proline 4-hydroxylases, which hydroxylate free l-proline to trans-4-hydroxy-l-proline, were screened in order to establish an industrial system for biotransformation of l-proline to trans-4-hydroxy-l-proline. Enzyme activities were detected in eight strains, including strains of Dactylosporangium spp. and Amycolatopsis spp. The Dactylosporangium sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library of Dactylosporangium sp. strain RH1 constructed in Escherichia coli. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29,715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in E. coli as a fused protein with the N-terminal 34 amino acids of the β-galactosidase α-fragment. The E. coli recombinant exhibited proline 4-hydroxylase activity that was 13.6-fold higher than the activity in the original strain, Dactylosporangium sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene.
PMCID: PMC99737  PMID: 10473412
25.  Glycosides of hydroxyproline: Some recent, unusual discoveries 
Glycobiology  2011;22(6):757-767.
Glycosides of hydroxyproline (Hyp) in the plant cell wall matrix were discovered by Lamport and co-workers in the 1960s. Since then, much has been learned about these Hyp-rich glycoproteins. The intent of this review was to compare and contrast some less common structural motifs, in nontraditional roles, to uncover themes. Arabinosylation of short-peptide plant hormones is essential for growth, cell differentiation and defense. In a very recent development, prolyl hydroxylase and arabinosyltransferase activity has been shown to have a direct impact on the growth of root hairs in Arabidopsis thaliana. Pollen allergens of mugwort and ragweed contain proline-rich domains that are hydroxylated and glycosylated and play a structural role. In the case of mugwort, this domain also presents a significant immunogenic epitope. Major crops, including tobacco and maize, have been used to express and produce recombinant proteins of mammalian origin. The risks of plant-imposed glycosylation are discussed. In unicellular eukaryotes, Skp1 (a subunit of the E3SCF ubiquitin ligase complex) harbors a key Hyp residue that is modified by a linear pentasaccharide. These modifications may be involved in sensing oxygen levels. A few studies have probed the impact of glycosylation on the structure of Hyp-containing peptides. These have necessarily looked at small, synthetic molecules, since natural peptides and proteins are often isolable in only minuscule amounts and/or are heterogeneous in nature. The characterization of native structural motifs, together with the determination of glycopeptide conformation and properties, holds the key to rationalizing nature's architectural design.
PMCID: PMC3336868  PMID: 22190471
glycosylation; hydroxyproline; peptide conformation

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